Project Summary CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) systems are RNA-based immune systems that function in prokaryotes to protect against viruses and other invasive mobile genetic elements. Our understanding of these adaptive immune systems function has increased exponentially in the last ten years. Importantly, this information has been exploited and harnessed into exceptionally powerful, novel research tools for genome editing and predictable control of gene expression with widespread industrial and medical applications. There are multiple diverse CRISPR-Cas systems that combat viruses by distinct mechanisms and with distinct machinery, each providing unique biology and potential applications to explore. CRISPR-Cas systems employ three general steps in invader defense: adaptation, crRNA, biogenesis, and invader silencing. The initial step results in integration of a fragment of invader DNA sequence into the CRISPR locus to provide a heritable record of the invasion and source of targeting information (adaptation). The CRISPR locus is transcribed and the transcript is processed to produce mature crRNA species (crRNA biogenesis). The crRNA and Cas proteins form an effector complex that seeks out and destroys the invading nucleic acid through base-pairing of the crRNA and destruction by an associated Cas protein nuclease (invader silencing). Translational research on CRISPR-Cas effector complexes has yielded the gene editing powerhouse CRISPR-Cas9 from the Type II-A system. The focus of my research is to understand invader silencing by the less well characterized Type III-A or Csm effector complex. Type III systems are unique in that they target RNA as well as DNA. The overall objective of this proposal is to obtain a detailed mechanistic understanding of how the RNA and DNA targeting activities of the Type II-A Csm system are achieved through the concerted interactions of the associated crRNA and six Cas proteins. Aim 1 is to elucidate the molecular basis of transcription-dependent DNA targeting by the Csm system. Aim 2 is to delineate the functional relationship of Csm6 with the Csm complex and its role in invader elimination. A powerful combination of mostly established in vitro and in vivo approaches will be employed to address the proposed specific aims. The studies will contribute to our fundamental understanding of CRISPR-Cas biology. In addition, the knowledge obtained will afford opportunities for the development of novel Type III CRISPR- Cas-based technologies with far reaching potential biotechnological and biomedical applications.